The aim of this study was to evaluate the inhibitory effect of antibiotics and Xiao'er Feire Kechuan Oral Solution on Mycoplasma pneumoniae (MP) clinical isolates.Twenty clinical isolates containing A-to-G transition at position 2063 and 10 clinical isolates without mutations in 23S rRNA V regions were randomly selected. The international standard strain FH was chosen as control strain. The minimum inhibitory concentration (MIC) of macrolide, quinolones, tetracycline, and Xiao'er Feire Kechuan Oral Solution to MP clinical isolates were performed using broth microdilution method.In vitro antibiotic susceptibility test of MP clinical isolates showed that MP showed high resistance to macrolide antibiotics (erythromycin and azithromycin); MIC of both were more than 64 μg/mL. The MICs of erythromycin and azithromycin for clinical isolates without mutations in 23S rRNA V regions were ≤0.5 μg/mL. The MICs of tetracycline and levofloxacin for all clinical isolated strains were ≤2.0 μg/mL and ≤1.0 μg/mL, respectively. The MIC of Xiao'er Feire Kechuan Oral Solution was 13.828∼6.914 mg/mL.In vitro, the drug resistance of MP to macrolide antibiotics is higher, MP clinical isolates are sensitive to tetracycline and levofloxacin, and Xiao'er Feire Kechuan Oral Solution also has a certain inhibitory effect on the macrolide-resistant MP.